Sudden death due to cardiac arrhythmias is one of the leading causes of death in the Western world. The most common disease responsible for sudden death is ischemic heart disease but in younger subjects inherited diseases such as hypertrophic cardiomyopathy and long QT syndrome are also important.
Cardiac arrhythmias may arise from abnormalities in impulse formation, impulse conduction, or a combination of both. The regulation of impulse formation and conduction involves a complex interaction between the autonomic nervous system, cardiac ion channels, and cardiac gap junctions.
The results of pharmacological prevention of especially ischemia-induced arrhythmias have been disappointing. Thus, clinical trials have documented that several class I and class III antiarrhythmic drugs increase mortality in patients with ischemic heart disease[1]. A common feature of all antiarrhythmic drugs presently in use is that they interfere with either cardiac ion channels (sodium, potassium, and calcium channels) or the autonomic nervous system, thereby interfering with the generation of the action potential. This is probably why they not only act antiarrhythmically, but also has a proarrhythmic action with the potential for inducing lethal arrhythmias particularly in patients with reduced left ventricular function, congestive heart failure or a history of sustained ventricular tachyarrhythmia. Examples of antiarrhythmic drugs are flecainide, encainide, moricizine, and quinidine. Antiarrhythmic drugs that lengthen cardiac repolarization such as amiodarone and sotalol are associated with potential development of a specific and striking arrhythmia, torsades de pointes. Torsades, a very fast ventricular arrhythmia, probably occurs when a set of associated features hypokalemia, bradycardia, and possibly delayed conduction alters membrane stability, promoting oscillations. Amiodarone, like sotalol, is approved only for life-threatening arrhythmias. The drug blocks the sodium channels and to some extent the calcium channels, and it also has beta-blocking effects. In early trials, side effects (which are dose-related) resulted in drug discontinuation in up to 20% of patients at one year. Cardiac toxicities include sinus bradycardia, atrioventricular block, congestive heart failure, and ventricular arrhythmias.
In summary, the currently available antiarrhythmic drugs have failed to prevent sudden death caused by cardiac arrhythmias. Therefore, there is a great unmet need for new, safe, and effective antiarrhythmic drugs in the treatment of life threatening arrhythmias. Due to the serious side effects that limit the use of the present antiarrhythmic drugs a new class of antiarrhythmic drugs with a completely different mode of action is desirable. As mentioned above the regulation of impulse formation and conduction is a complex interaction between the autonomic nervous system, the cardiac ion channels, and the cardiac gap junctions. Hitherto the development of antiarrhythmic drugs has focused on the autonomic nervous system and the cardiac ion channels and no currently available drugs function as cardiac gap junction openers. However, recently several lines of evidence have proven the important role for gap junctions in the development of arrhythmias and the modulation of gap junctions is therefore a very interesting new target in the treatment of arrhythmias.
Gap junctions are specialized regions of the cell membrane with clusters of hundreds to thousands of densely packed gap junction channels that directly connect the cytoplasmic compartment of two neighboring cells. The gap junction channels are composed of two hemichannels (connexons) provided by each of two neighboring cells. Each connexon consists of six proteins called connexins. The connexins are a large family of proteins all sharing the basic structure of four transmembrane domains, two extracellular loops, and a cytoplasmic loop. There is a high degree of conservation of the extracellular loops and transmembrane domains among species and connexin isoforms. The length of the C-terminus, however, varies considerably giving rise to the classification of the connexins on the basis of the molecular weight. The distribution of the different types of connexins (Cx) varies throughout the heart. The Cx43 isoform is the predominant type in the ventricles whereas Cx40 is the must abundant isoform in the atrias and the conduction system. The gap junction channel can switch between an open and a closed state by a twisting motion. In the open state ions and small molecules smaller than about 1000 D can pass through the pore. The conduction of the electrical impulse takes place through the gap junctions and normally functioning gap junctions are therefore a prerequisite for normal conduction and thereby normal rhythm of the heart.
An increased understanding of the important role of gap junctions in abnormal conduction has been provided by the development of knockout mice lacking different types of connexins. From these studies it has been shown that mice homozygous for a targeted deletion of the Cx43 gene die shortly after birth from cardiac and pulmonary malformations, whereas heterozygous mice survive. However, the heterozygous genotype has a significantly slowed conduction compared to wild-type mice[2]. In adult mice (6-9 month old) ventricular epicardial conduction of paced beats is slowed by 44% and QRS complexes of ECG recordings are significantly prolonged compared to those of wild-type mice. The reduced expression of Cx43 is directly linked to an increased incidence of ventricular arrhythmias during ischemia in mice heterozygous for the Cx43 gene deletion[3]. Thus, the incidence of spontaneous ventricular tachycardia after the induction of regional ischemia in isolated perfused hearts from heterozygous mice is twice the incidence in wild-type hearts. In addition, mice with cardiac specific loss of Cx43 develop spontaneous ventricular arrhythmias and sudden cardiac death, with 100% mortality by two months of age. Knockout of the Cx40 gene is not fatal, however, atrial, atrioventricular, and His-Purkinje conduction are significantly slower in Cx40−/− mice relative to Cx40+/+ mice, and Cx40−/− mice are at increased risk of arrhythmias and bundle branch block[4-6].
The link between abnormalities in connexins and heart disease has also been established in humans. One example is Chagas' disease caused by the protozoan parasite Trypanosoma cruzi. This disease is a major cause of cardiac dysfunction in Latin America. An altered Cx43 distribution has been observed in cells infected by Trypanosoma cruzi and this alteration may be involved in the genesis of the conduction disturbances characterizing the disease[7]. Several studies of the expression and distribution of Cx43 in chronically ischemic, hibernating, or hypertrophied hearts also describe a reduced degree of Cx43 expression and a changed pattern of distribution[8-10]. In fact the expression and/or distribution of connexins have been altered in all chronic disease states of the heart investigated so far.
In summary, there is plenty of evidence linking malfunction or absence of gap junctions to an increased risk of arrhythmias and plenty of evidence showing an altered connexin expression/distribution in chronic heart disease. As mentioned above no currently available antiarrhythmic drugs act by increasing gap junction function. However a group of peptides (the antiarrhythmic peptides) capable of increasing gap junction conductance has been described in the past.
The Antiarrhythmic Peptides
In 1980, a hexapeptide with a molecular weight of 470D was isolated from bovine atria by Aonuma and colleagues[11]. In neonatal rat cardiomyocytes, it was demonstrated that 0.1 μg/ml of this peptide could convert fibrillation induced by either ouabain, high calcium (3 mM) or low potassium (0.7 mM) to normal rhythm. In addition, 2.5-5.0 μg/ml of this peptide could convert arrhythmic movement of isolated rat atria induced by the combination of low potassium (0.3 mM) and acetylcholine to normal rhythm. Thus, this peptide was named antiarrhytmic peptide (AAP) (Comparative Example 1 below (CE1)). When added to cell culture medium, AAP increased the number of beating centers, the relative content of spreading cells and protein synthesis[12]. In 1982, the amino acid sequence of AAP was determined to be (SEQ ID NO: 1) H-Gly-Pro-4Hyp-Gly-Ala-Gly-OH[13]. In later in vivo studies, the antiarrhythmic effect of AAP observed in vitro was confirmed. It was shown that AAP, 10 mg/kg, was effective against CaCl2—, oubain and acotinine-induced arrhythmia in mice[14]. Several synthetic derivatives of AAP have been tested and found to be more potent than the endogenous AAP against experimentally induced arrhythmias in mice and rats[15-17]. The synthetic derivative that has been most thoroughly investigated is AAP10 (SEQ ID NO: 2) (H-Gly-Ala-Gly-4Hyp-Pro-Tyr-NH2) (Comparative Example 2 below (CE2)). In the isolated perfused rabbit heart 0.1 nmol/l to 10 nmol/l of this peptide reduced the dispersion of activation-recovery intervals measured at 256 ventricular epicardial electrodes during normal conditions[18]. AAP10 had no effect on mean action potential duration, left ventricular end-diastolic pressure, coronary flow, QRS duration, or on the PQ interval. If hearts were subjected to regional ischemia by occlusion of the descending branch of the left coronary artery for 30 min, pretreatment with 10 nmol/l AAP10 led to a significant reduction in ischemia-induced alterations of activation patterns and reduced dispersion of activation-recovery intervals[18]. Additional studies showed that AAP10 did not affect the action potential in isolated papillary muscles from guinea pig hearts in concentrations up to 1 μmol/l[18]. These findings are in accordance with the findings of Argentieri and colleagues[19] who investigated the mechanism of the antiarrhythmic properties of AAP by examining the effect on the action potential in isolated canine purkinje fibers. In this model, AAP did not affect inotropy or any of the eletrophysiological parameters measured (maximum diastolic potential, action potential amplitude, maximum rate of depolarization, and action potential duration at 50% and 95% repolarization). Therefore, it was concluded that AAP's does not affect transmembrane ion currents. In guinea pig papillary muscle the effect on coupling time, i.e. the time interval between electrostimulation and onset of the action potential, was examined[20]. It was found that high concentrations of AAP10 (1 μM) could decrease the stimulus-response-interval by about 10% under normoxic conditions. Furthermore, during hypoxia and glucose-free perfusion the increase in stimulus-response-interval indicating uncoupling was prevented by 10 nmol/l of AAP10. Since the effect of AAP10 on coupling time was most pronounced on poorly coupled cells, the authors suggested that AAP10 preferentially acts on poorly coupled cells. The effect on coupling time suggested that AAP10 exerts its actions via an effect on gap junction conductance. To test this theory, the authors examined the effect of AAP10 on gap junction conductance in adult guinea pig ventricular cardiomyocytes using the double-cell voltage clamp technique. These studies demonstrated that 10 nmol/l AAP10 produced a rapid and reversible increase in gap junction conductance. Thus, the antiarrhythmic properties of AAP10 were explained by an improvement of gap junction coupling thereby reducing action potential dispersion and preventing slowing of conduction.
In summary, the antiarrhythmic peptides are a group of peptides that exert their effect selectively on gap junctions and thus decrease cellular uncoupling and reduce dispersion of action potential duration and refractoriness without affecting the action potential duration or shape. Therefore, the antiarrhythmic peptides are expected to lack the proarrhythmic effects limiting the use of many currently available antiarrhythmic drugs. This makes the antiarrhythmic peptides extremely interesting as a potentially new and safer class of antiarrhythmic compounds. However, the native AAP as well as the synthetic AAP10 possess several undesired features, such as, low stability, high effective concentration etc. that has hitherto prevented their utilisation as drugs. Grover and Dhein[21] have characterised two semi cyclic conformations of AAP10 using nuclear magnetic resonance spectroscopy. Therefore, one approach to obtaining a stable antiarrhytmic peptide could be the provision of cyclic derivatives of antiarrhythmic peptides. DE19707854 discloses apparently cyclic (SEQ ID NO: 3) CF3C(OH)-Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH and cyclic (SEQ ID NO: 4) CO-Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH having the same antiarrhythmic properties as AAP and AAP10, but stated to have improved stability in aqueous solution and after repeated cycles of freezing and thawing. However, the experimental conditions described in DE19707854 are insufficient for the preparation of said cyclic compounds, and the chemical identification data given therein using HPLC is not sufficient for identification of said cyclic compounds. U.S. Pat. No. 4,775,743 discloses HP5, a peptide derivative having the sequence (SEQ ID NO: 5) N-3-(4-hydroxyphenyl)propionyl-Pro-4Hyp-Gly-Ala-Gly-OH and being active against platelet agglutination. Dhein and Tudyka[22] have reviewed the literature on peptides including peptide derivatives belonging to the group of antiarrhythmic peptides for activity and concentration, cf. table 1 therein, and found only 7 compounds to be active and further 4 compounds to be weakly active. However, none of these peptides or peptide derivatives have been shown to be sufficiently stable to be effective in a therapy regimen.
Furthermore, cyclic depsipeptides having antiarrhythmic action but having an ester bond being labile towards endogenous esterases are disclosed in JP patent application No. 08281636 and in JP patent application No. 09308589. Moreover, WO96/21674 discloses AAP10 derivatives where a hydrogen at the phenyl ring of the tyrosine residue has been substituted with halogen. Said AAP10 derivatives have antiarrhythmic properties and a reduced proarrhythmic risk compared to lidocain and flecainid.
The following AAP peptides and AAP-like compounds are described in the literature:
(AAP) H-Gly-Pro-4Hyp-Gly-Ala-Gly-(SEQ ID NO: 6) OH, H-Gly-Pro-4Hyp-OH, H-Gly-Pro-OH, H-Gly-Pro-Leu-OH, H-Gly-Pro-4Hyp-Gly-OH, H-Gly-Pro-Leu-Gly-Pro-OH,(SEQ ID NO: 7) H-4Hyp-Gly-OH, H-Gly-Ala-Gly-OH, H-Gly-Gly-Gly-OH, H-Pro-Pro-Gly-OH, H-Pro-4Hyp-Gly-Ala-Gly-OH,(SEQ ID NO: 8) H-Pro-4Hyp-OH, H-Pro-4Hyp-Gly-OH, H-Pro-4Hyp-Gly-Ala-OH, (HP5) N-3-(4-hydroxyphenyl)pro-(SEQ ID NO: 9) pionyl-Pro-4Hyp-Gly-Ala-Gly-OH, N-3-phenylpropionyl-Pro-4Hyp-Gly-(SEQ ID NO: 10) Ala-Gly-OH, N-3-phenylpropyl-Pro-4Hyp-Gly-Ala-(SEQ ID NO: 11) Gly-OH, N-3-(4-hydroxyphenyl)propionyl- Pro-4Hyp-Gly-Ala-OH, N-3-(4-hydroxyphenyl)propionyl- Pro-4Hyp-Gly-OH, N-3-(4-hydroxyphenyl)propionyl- Pro-4Hyp-OH, N-3-(4-hydroxyphenyl)propionyl-(SEQ ID NO: 12) Pro-Pro-Gly-Ala-Gly-OH, (AAP10) H-Gly-Ala-Gly-4Hyp-Pro-(SEQ ID NO: 13) Tyr-NH2, H-Gly-Ala-Gly-4Hyp-Pro-Tyr-OH,(SEQ ID NO: 14) H-Ala-Gly-4Hyp-Pro-Tyr-NH2,(SEQ ID NO: 15) H-Gly-Sar-Pro-Gly-Ala-Gly-OH,(SEQ ID NO: 16) H-Gly-Pro-Sar-Gly-Ala-Gly-OH,(SEQ ID NO: 17) H-Gly-Sar-Sar-Gly-Ala-Gly-OH,(SEQ ID NO: 18) H-Gly-Ala-Gly-Hyp-Pro-Tyr(3-I)-NH2(SEQ ID NO: 19) H-Gly-Ala-Gly-Hyp-Pro-Tyr(3-F)-NH2(SEQ ID NO: 20) H-Gly-Ala-Gly-Hyp-Pro-Tyr(3-Cl)-(SEQ ID NO: 21) NH2 H-Gly-Ala-Gly-Hyp-Pro-Tyr(3-Br)-(SEQ ID NO: 22) NH2 H-Arg-Ala-Gly-Hyp-Pro-Tyr-NH2(SEQ ID NO: 23) H-Val-Ala-Gly-Hyp-Pro-Tyr-NH2(SEQ ID NO: 24) H-Ala-Ala-Gly-Hyp-Pro-Tyr-NH2(SEQ ID NO: 25) H-Gly-Ala-Gly-Hyp-His-Tyr-NH2(SEQ ID NO: 26) H-Gly-Ala-Gly-Hyp-Pro-Phe-NH2(SEQ ID NO: 27) Cyclo(CF3C(OH)-Gly-Ala-Gly-4Hyp-(SEQ ID NO: 28) Pro-Tyr-CONH), and Cyclo(CO-Gly-Ala-Gly-4Hyp-Pro-Tyr-(SEQ ID NO: 29) CONH).
The following compounds
H-Gly-Pro-4Hyp-Gly-Ala-Gly-OH(SEQ ID NO: 30) (AAP), H-Gly-Pro-4Hyp-Gly-Ala-Gly-OH,(SEQ ID NO: 31) H-Gly-Ala-Gly-4Hyp-Pro-Tyr-NH2(SEQ ID NO: 32) (AAP10), H-Gly-Ala-Gly-4Hyp-Pro-Tyr-OH,(SEQ ID NO: 33) H-Gly-Ala-Gly-Hyp-Pro-Tyr(3-I)-(SEQ ID NO: 34) NH2, H-Gly-Pro-Sar-Gly-Ala-Gly-OH,(SEQ ID NO: 35) N-3-(4-hydroxyphenyl)propionyl-(SEQ ID NO: 36) Pro-4Hyp-Gly-Ala-Gly-OH (HP5), N-3-phenylpropionyl-Pro-4Hyp-Gly-(SEQ ID NO: 37) Ala-Gly-OH, N-3-(4-hydroxyphenyl)propionyl-(SEQ ID NO: 38) Pro-Pro-Gly-Ala-Gly-OH, Cyclo(CF3C(OH)-Gly-Ala-Gly-4Hyp-(SEQ ID NO: 39) Pro-Tyr-CONH), and Cyclo(CO-Gly-Ala-Gly-4Hyp-Pro-Tyr-(SEQ ID NO: 40) CONH)have shown activity or weak activity in test models, cf., e.g., Dhein and Tyduka (1995).
Although active antiarrhythmic peptides have been provided none of these have lead to the development of a much sought for antiarrhythmic medicament. The purpose of the present invention is to provide further antiarrhythmic peptides and functional analogues thereof useful in the treatment of various coronary heart diseases and useful for the preparation of medicaments. Furthermore, the novel peptides herein increase gap junction intercellular communication (GJIC) in vertebrate tissue, and specifically in mammalian tissue, and are useful in the treatment of a wide spectrum of diseases and ailments in vertebrates, such as mammals, relating to or caused by a decreased function of intercellular gap junction communication as is described below.